Apoptosis may be a common mechanism of immune-system mediated B-cell death in insulin-dependent diabetes mellitus (IDDM) and an abnormally elevated concentration of intracellular free calcium, [Ca2+]i can be part of the apoptotic pathway, perhaps even triggering apoptosis. An earlier study (Rhoten and Sergeev, 1994) showed that clonal rat B-cells with an increased content of the high affinity calcium-binding protein calbindin D28k maintained much more effectively than control cells a normal [Ca2+]i when challenged with potentially apoptosis-producing increases in [Ca2+]i. Therefore, calbindin D28k might rectify immune-system-mediated increases in [Ca2+]i to inhibit apoptosis and thus preserve B-cell function. The goals of this project are: 1) determine the role of constitutively expressed calbindin D28k in modulating apoptosis in B-cells and, 2) to determine how different levels of calbindin D28k influence Ca2+ regulatory mechanisms and insulin secretion. The hypothesis to be tested is that normal pancreatic B-cells with high endogenous levels of calbindin D28k have a lower basal rate of apoptosis and are more resistant to pro-apoptotic agent-mediated apoptosis than B-cells with low levels of calbindin-D28k. To test this hypothesis a very appropriate model, the chicken B-cell which has a high endogenous calbindin D28k content will be studied. The investigators will determine basal and pro-apoptotic agent-mediated rates of apoptosis in normal and experimentally calbindin D28k depleted chicken B-cells, and in rat B-cells. The proapoptotic agents which increase [Ca2+]i will be ionomycin and thapsigargin. Apoptosis will be assessed in situ by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling method. Cytofectin-mediated uptake of C5 propynyl pyrimidine phosphorothioate antisense oligonucleotides and a dietary method will be used to reduce the high endogenous levels of calbindin D28k. Western blotting and ELISA will be used to validate anticipated levels of calbindin D28k and to evaluate levels of apoptosis regulating (BCL-2, PI-3 kinase) and control (calbindin D9k, calmodulin) proteins. The investigators will determine the effect of depleting calbindin D28k on intracellular Ca2+ responses, insulin secretion, and the cellular localization of calbindin D28k and insulin. Fluorescent ratiometric in situ video imaging and Ca2+ indicator fura-2 will be used to evaluate changes in Ca2+ responses. Insulin secretion will be measured by ELISA. Calbindin-D28k and insulin will be localized three-dimensionally by confocal microscopy. The effect of TNFa, a cytokine up-regulator of gene expression of calbindin D28k, on apoptosis, calbindin-D28k content and B-cell function will be determined. The long term goal is to integrate calbindin D28k gene expression into a therapeutic strategy that will protect the B-cell from immune-system-mediated destruction and prevent IDDM.